2.1. Synthesis and Characterization of Gold Nanoparticles
In the present investigation, AuNPs were formed by simply mixing astaxanthin with gold chloride solution without any external energy. The nanoparticles represented hereafter will be referred to as astaxanthin-reduced gold nanoparticles (Atx-AuNPs). Astaxanthin pigment has two terminal ring systems connected by double bonds of carbohydrate and displays strong antioxidant and anticancer properties [
12]; in addition, it can act as a reducing biomolecule. Phytochemical-reduced AuNPs have great biocompatibility with promising anticancer effect and other biological applications such as biosensing, PTT, and PAT imaging [
13]. A significant color change was observed within 40 min, and a surface plasmon resonance (SPR) band appeared at 534 nm and 985 nm (
Figure 1A). Similarly, Klekotko reported the presence of one narrow SPR at 540 nm for spherical and one broad SPR around 900–1000 nm for anisotropic shapes like triangular and hexagonal AuNPs by green reduction using mint extract [
14]. We are the first to report the synthesis of AuNPs using astaxanthin as a reducing agent. The appearance of optical response broad band around 985 nm in NIR was interesting, and nanoparticles of NIR absorption have potential applications in PTT, PDT, and PAT technology [
15]. PAT, PDT, and PTT using NIR have the unique advantage of low systemic toxicity, remote controllability, and being able to image cancer cells in deeply situated tissue [
16]. The transmission electron microscopy (TEM) image (
Figure 1C) shows the presence of two major shapes of crystalline nanoparticles, spherical and triangular, which are corroborated by two SPR peaks. The sharp band at 534 nm and broad band around 985 nm correspond to spherical and anisotropic shapes respectively and size of the nanoparticles found to be in broad range from 30 to 250 nm. Based on dynamic light scattering (DLS) analysis, most of the particles’ size falls in the range of 60–120 nm (
Figure 1B). Anisotropic shapes of nanoparticles includes majorly triangle found in
Figure 1C may be responsible for the shift of UV-Vis spectrum absorption around 985 nm.
The SPR band is influenced by size, shape, morphology, and its interacting medium [
17]. The present result shows the formation of a broad range of nanoparticles. It should be noted that the biological molecules used in nanoparticle synthesis process can also influence the SPR of bioreduced AuNPs [
13,
18,
19]. Many studies manifested rapid synthesis of AuNPs using biological materials [
18] having good biocompatibility with promising biological activities like antimicrobial and anticancer effects [
19].
FTIR analysis was performed to find the possible biomolecules of astaxanthin responsible for bioreduction or coating of Atx-AuNPs. Similar functional groups were found between interferograms of astaxanthin and Atx-AuNPs (
Figure 2A). A strong band observed at 1071 cm
−1 in astaxanthin and 1014 cm
−1 in Atx-AuNPs is characteristic of –CH=CH
2– bending, and the carbohydrate stretch acts as linker for the two aromatic rings in astaxanthin. A C–C stretch of aromatic rings was found at 1455 cm
−1 and 1409 cm
−1 in astaxanthin and Atx-AuNPs, respectively. The presence of aromatic functional groups in Atx-AuNPs was additionally confirmed by the peak at 765 cm
−1, and the same group was found in astaxanthin at 861 cm
−1. The peak at 1722 cm
−1 in Atx-AuNPs indicated the involvement of carbonyl group in nanoparticle reduction. According to existing reports, carbonyl, hydroxyl, carbohydrate [
20], and aromatic hydrocarbon [
21] can reduce and cap AuNPs.
The X-ray diffraction (XRD) pattern further confirmed the presence of gold particles (
Figure 2B). The intensities of crystalline AuNPs were recorded in XRD from 20° to 80°. The intense diffraction peaks at 2θ of 38.26°, 44.60°, 64.67°, and 77.54° corresponded to (111), (200), (220), and (311), respectively, and the pattern agreed well with the standard (JCPDS No. 04-0784) and earlier reports [
22]. The peak assigned to (111) was stronger than the rest of the peaks.
2.2. Assessment of Cytotoxic Effect of Atx-AuNPs
To assess the cytotoxic effect of synthesized Atx-AuNPs against Human breast cancer cell line (MDA-MB-231 cells), 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was performed with different concentrations ranging from 10 to 100 µg·mL
−1 for 24 h. Atx-AuNPs showed effective antiproliferative effects with increasing concentration (
Figure 3), and 50% inhibitory concentration was found as 50 µg·mL
−1. They inhibited almost 60% of cell growth above a concentration of 80 µg·mL
−1; however, they do not show much variation within the concentration range of 80–100 µg·mL
−1. The cell viability increased when the doses decreased. The cytotoxic effect of astaxanthin is low when compared with Atx-AuNPs.
The use of AuNPs has increased significantly in medical applications. Previous studies reported that the cytotoxic effect of AuNPs will vary depend on concentration [
23], synthesis methodology [
24], and types of cell [
25]. Generally AuNPs induce significant cytotoxicity in concentrations above 100 µg·mL
−1. El-Kassas reported 80% of Michigan Cancer Foundation-7 cell viability at a 100 µg·mL
−1 concentration of AuNPs [
26]. In the present study we used 100 µg·mL
−1 of Atx-AuNPs as the maximum concentration, which exerts 68% cell death.
2.3. Microscopic Analysis of Cell Death
Bright field microscopic images show the difference between controls and treated cells. We can observe morphological changes like disturbed cell shape, growth inhibition, and cytoplasmic condensation in Atx-AuNPs-treated cells (
Figure 4A), which are not seen in the control photograph; the cells remain live with a uniform structure. Atx-AuNPs-induced morphological alteration was observed using nucleic acid binding Acridine orange-Ethidium bromide (AO-EB) staining. Control cells appeared in a uniformly light green color. As control live cells exclude orange EB stain, greenish-yellow cells were not documented in control cells. Cells were very different in the treatment group compared to the control cells.
Figure 4B showed cell shrinkage and nuclei condensation, which is a step of apoptosis. Clearly fragmented nuclei (arrow marked) were observed and cells were shrunken in the treatment groups. Some bulged necrotic cells were also observed (dashed arrows). AO-EB fluorescent staining allowed us to discriminate Atx-AuNPs-induced apoptotic cells from control cells.
We also used the following nuclei stains to differentiate cell death. Hoechst stain stains the nuclei of the cells regardless of their viability [
27], allowing one to distinguish whether or not Atx-AuNPs caused the changes in nuclei morphology. Atx-AuNPs-induced nuclei condensation was photographed and represented by arrows in the treated cells (
Figure 4C). The number of abbreviated and cleaved nuclei increased in the 50 µg·mL
−1 dose treatment. The photograph of control cells shows an absence of punctate nuclei, and expressed cells remain normal.
Propidium iodide is a widely used nuclei staining that can enter into cells depending on the plasma membrane integrity, and therefore cannot stain living and early apoptotic cells [
28,
29]. Results from the PI staining indicate that the hallmark event of apoptosis, karyorrhexis (condensation of chromatin until it breaks into the cell) [
30], was accelerated by Atx-AuNPs treatment (
Figure 4D), which supports the results of the Hoechst staining. A round, condensed nucleus with red fluorescence was observed in nanoparticle-treated cells, indicated by arrows, and the nuclei of control cells remained unstained as PI cannot permeate the plasma membrane of viable cells.
2.4. Photoacoustic Image
Tissue-mimicking phantom (control) and Atx-AuNP-treated cells are shown in
Figure 5B. The maximum intensity projection (MIP) image along the Z-axis to the XY plane of the phantom is displayed over an 18 mm × 8 mm field of view (
Figure 5C). We can observe the photoacoustic signal-generated image of Atx-AuNPs-treated cells; at the same time, control sample cells were not detected (
Figure 5C). High-amplitude photoacoustic signals were detected from the inclusions of treated cells. The incident light was homogeneously distributed over the volume of cell inclusions, as observed in a 3 dimentional image (
Figure 5D), which confirms the optical scattering property of Atx-AuNPs inside the cells.
Figure 5D shows the 3D image of the phantom with an 18 mm × 8 mm × 6 mm field of view. Atx-AuNPs act as acoustic scatter, and a gelatin-based phantom provides the desirability of soft tissue. The scattered NIR results in hyperthermic expansion in the treated cells, which generates a broadband signal. Then, the signal was received by an ultrasound transducer to produce an image (
Figure 5A). The gelatin-based phantom used here mimics the electromagnetic properties of biological tissue; this kind of
in vitro phantom is rapidly emerging as an imaging technology used by many research groups [
13]. This study shows that astaxanthin-synthesized nanoparticles can be used for photoacoustic imaging.